Epigenetic influences of low-dose bisphenol A in primary human breast epithelial cells

https://doi.org/10.1016/j.taap.2010.07.014Get rights and content

Abstract

Substantial evidence indicates that exposure to bisphenol A (BPA) during early development may increase breast cancer risk later in life. The changes may persist into puberty and adulthood, suggesting an epigenetic process being imposed in differentiated breast epithelial cells. The molecular mechanisms by which early memory of BPA exposure is imprinted in breast progenitor cells and then passed onto their epithelial progeny are not well understood. The aim of this study was to examine epigenetic changes in breast epithelial cells treated with low-dose BPA. We also investigated the effect of BPA on the ERα signaling pathway and global gene expression profiles. Compared to control cells, nuclear internalization of ERα was observed in epithelial cells preexposed to BPA. We identified 170 genes with similar expression changes in response to BPA. Functional analysis confirms that gene suppression was mediated in part through an ERα-dependent pathway. As a result of exposure to BPA or other estrogen-like chemicals, the expression of lysosomal-associated membrane protein 3 (LAMP3) became epigenetically silenced in breast epithelial cells. Furthermore, increased DNA methylation in the LAMP3 CpG island was this repressive mark preferentially occurred in ERα-positive breast tumors. These results suggest that the in vitro system developed in our laboratory is a valuable tool for exposure studies of BPA and other xenoestrogens in human cells. Individual and geographical differences may contribute to altered patterns of gene expression and DNA methylation in susceptible loci. Combination of our exposure model with epigenetic analysis and other biochemical assays can give insight into the heritable effect of low-dose BPA in human cells.

Introduction

Bisphenol A (BPA), first synthesized by A. P. Dianin in 1891, has been widely used as a cross-linking reagent in the manufacture of epoxy resins since 1950s (Vogel, 2009). It is extensively used in a board range of products, including toys, water pipes, drinking bottles, baby bottles, food containers, tubing, and dental sealants (Welshons et al., 2006). Presently, the worldwide production of BPA exceeds 3 billion kg/year (Vandenberg et al., 2009). Studies have shown that BPA can be released from incomplete polymerization upon heating or leached out through normal use (Mountfort et al., 1997, Kang et al., 2003, Goodson et al., 2004). Because of its ubiquity in environment, low levels of BPA can be detected in 92.6% of urine samples (≥ 6 years of age ranging from 0.4 to 149 μg/l) in the National Health and Nutrition Examination Survey (NHANES) 2003–2004 (Calafat et al., 2008, CDC, 2009). Animal studies have shown that these low levels of BPA exposure may alter developmental programs of sensitive end organs, like mammary and prostate gland, during critical stages of early development (Markey et al., 2001, Nikaido et al., 2004, Timms et al., 2005). The changes may persist into puberty and adulthood, suggesting an imprinting process being imposed in differentiated breast epithelial cells (Markey et al., 2001, Munoz-de-Toro et al., 2005).

The molecular mechanisms by which early memory of BPA exposures can be imprinted in breast progenitor cells and then passed onto their epithelial progeny are not well understood. One distinct possibility is through epigenetic remodeling of DNA structure without altering the nucleotide sequence itself. The changes, including DNA methylation, frequently occur in GC-rich promoter CpG islands of transcriptionally repressed genes (Ohm and Baylin, 2007, Widschwendter et al., 2007). Studies have suggested that DNA methylation of a promoter CpG island, or promoter hypermethylation, can be initiated in progenitor genomes and heritably passed onto the differentiated progeny (Jones and Baylin, 2007, Marotta and Polyak, 2009). This epigenetic process is known to cause phenotypic variations among individuals and contributes to the development of pathological conditions, like cancer (Feinberg et al., 2006, Esteller, 2007).

Previous studies of epigenetic effects of BPA preexposure mainly rely on animal models and epidemiological surveys (Ho et al., 2006, Dolinoy et al., 2007, Prins et al., 2008, Yaoi et al., 2008). Whereas these observations strongly implicate that the exposure to low-dose BPA is potentially harmful to human health, the challenge encountered is validation studies of the findings in primary human cells. In this regard, we have recently established a human preexposure model for epigenetic studies (Cheng et al., 2008, Hsu et al., 2009). In the model, breast progenitor cells were first exposed to different environmental chemicals, and then these cells were differentiated into epithelial cells in the absence of these environmental stimulants. We hypothesize that slow-dividing progenitor cells have a longer life span and thus are more susceptible to environmental injuries and can transmit this injured memory to their differentiated progeny through epigenetic mechanisms. In our previous studies, the preexposure to 17β-estradiol (E2) and diethylstilbestrol (DES) may trigger epigenetic repression of protein-coding genes and non-coding microRNAs, some of which exhibit promoter hypermethylation in breast cancer cells (Cheng et al., 2008, Hsu et al., 2009).

Here, we extended this preexposure study to evaluate epigenetic effects of low-dose BPA in human breast epithelial cells. As a result of chronic exposure to BPA, activation of estrogen receptor α (ERα)-mediated signaling and subsequent alterations of responsive gene expression were observed in the differentiated epithelial progeny. This heritable influence on gene expression was similarly observed in ERα-positive breast tumors.

Section snippets

Tissue samples and cell culture

Breast tissues, obtained from individuals undergoing mastectomy or reduction mammoplasty, were collected in accordance with the protocols approved by the Institutional Review Boards of the Ohio State University and the National Taiwan University Hospital. For isolation of breast progenitor cells, non-cancerous tissues (age: 17–42 years old) were enzymatically dissociated via collagenase digestion as described previously (Hsu et al., 2009). Single cells were grown into floating spherical colonies

Effect of low-dose BPA on the nuclear localization of ERα in MDECs

Environmental chemicals, such as BPA, are known to act as estrogenic ligands that activate or deactivate gene transcription in breast epithelial cells (Soto et al., 2006, Dairkee et al., 2008). To determine whether BPA causes an estrogen-like effect, we performed immunofluorescence analyses in MDECs (un-exposed) transiently treated with different doses (ranges: 1–1,000 nM) of BPA at 0, 5, 30, 60, and 120 min (Fig. 1A). BPA, as a weak estrogenic ligand, caused maximized ERα internalization at 30 

Discussion

When acutely exposed to estrogenic ligands, signal transduction is mediated in part through nuclear hormone receptors, such as ERα (Bjornstrom and Sjoberg, 2005). We have previously shown that the hallmark of this signal transduction is the translocation of cytoplasmic ERα into the nucleus of a normal breast epithelial cell (Hsu et al., 2009). Unlike E2 and DES, BPA is considered to be a weak estrogenic ligand based on the present immunofluorescence analysis and previous receptor binding assays

Conclusions

In the present study, we have shown that the mammosphere exposure system is a valuable tool for validation studies of BPA findings based on animal models. We observed heritable effects of low-dose BPA on the nuclear localization of ERα and differential gene expression in primary MDECs. Long-term exposure of breast progenitor cells to BPA may promote ligand-independent ERα actions in differentiated progeny. Furthermore, genetic variations of individuals may contribute to differential

Conflict of interest statement

All authors declare that they have no competing financial interests.

Acknowledgments

This work was supported by the National Institutes of Health [U01 ES015986, R01 CA069065, and R01 ES017594 to T.H.-M.H]. We thank Jeff Apostolos for his technical assistance.

References (47)

  • Y. Nikaido et al.

    Effects of maternal xenoestrogen exposure on development of the reproductive tract and mammary gland in female CD-1 mouse offspring

    Reprod. Toxicol.

    (2004)
  • G.S. Prins et al.

    Developmental exposure to bisphenol A increases prostate cancer susceptibility in adult rats: epigenetic mode of action is implicated

    Fertil. Steril.

    (2008)
  • C.A. Richter et al.

    In vivo effects of bisphenol A in laboratory rodent studies

    Reprod. Toxicol.

    (2007)
  • E. Rohrdanz et al.

    The phytoestrogen daidzein affects the antioxidant enzyme system of rat hepatoma H4IIE cells

    J. Nutr.

    (2002)
  • A.M. Soto et al.

    Strengths and weaknesses of in vitro assays for estrogenic and androgenic activity

    Best Pract. Res. Clin. Endocrinol. Metab.

    (2006)
  • T. Vaissiere et al.

    Epigenetic interplay between histone modifications and DNA methylation in gene silencing

    Mutat. Res.

    (2008)
  • T. Yaoi et al.

    Genome-wide analysis of epigenomic alterations in fetal mouse forebrain after exposure to low doses of bisphenol A

    Biochem. Biophys. Res. Commun.

    (2008)
  • L. Bjornstrom et al.

    Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on target genes

    Mol. Endocrinol.

    (2005)
  • H. Buteau-Lozano et al.

    Xenoestrogens modulate vascular endothelial growth factor secretion in breast cancer cells through an estrogen receptor-dependent mechanism

    J. Endocrinol.

    (2008)
  • A.M. Calafat et al.

    Exposure of the U.S. population to bisphenol A and 4-tertiary-octylphenol: 2003–2004

    Environ. Health Perspect.

    (2008)
  • CDC

    Fourth National Report on Human Exposure to Environmental Chemicals

    (2009)
  • A.S. Cheng et al.

    Epithelial progeny of estrogen-exposed breast progenitor cells display a cancer-like methylome

    Cancer Res.

    (2008)
  • S.H. Dairkee et al.

    Bisphenol A induces a profile of tumor aggressiveness in high-risk cells from breast cancer patients

    Cancer Res.

    (2008)
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